AID	Name	Type	Comment
602183	qHTS for Inhibitors of mutant isocitrate dehydrogenase 1 (IDH1): Summary	summary	Summary AID

Malignant glioblastomas (WHO grade IV), including primary and secondary glioblastomas, are among the most lethal with a median survival of one year, and unfortunately they are also the most prevalent type of brain tumors [1]. By and large the standard of care of gliomas remains the use of the oral alkylating agent temozolomide and radiotherapy following surgical tumor resection [2]. There is an urgent unmet medical need for novel therapeutics for gliomas.

IDH mutations occur in up to 70% of grade II-IV secondary glioblastomas, and IDH1 R132H is the most prevalent mutation [3]. Interestingly, IDH mutations are also present in ~10% of AML (acute myeloid leukemia) patients [4]. Wild-type IDH1 catalyzes the conversion of isocitrate to -ketoglutarate with the concomitant reduction of NADP+ to NADPH. In contrast, IDH1 R132H catalyzes the conversion of -ketoglutarate to 2-hydroxyglutarate (2-HG) with the concomitant oxidation of NADPH to NADP+ [5]. Recently, a second significant advancement for the field was the finding that IDH is an oncogene, and that its metabolic product 2-HG may contribute to the pathogenesis of IDH-mutated cancers. Indeed, unbiased metabolite profiling of a U87MG stable cell line engineered to express IDH1 R132H mutant protein demonstrated that mutated IDH1 confers a gain-of-function to produce the onco-metabolite 2-HG, and in effect classifying IDH1 as an oncogene [5]. These studies demonstrated the power of utilizing 2-HG as a biomarker for IDH-mutated cancers, as well as adding to the wealth of data linking the metabolite 2-HG to cancer. Although this link to cancer is tantalizing, the direct evidence of how 2-HG drives pathogenesis of gliomas and AML remains a subject of further research.

The goal of this screen is to find small-molecule inhibitors of IDH1 R132H using a biochemical assay that measures substrate NADPH consumption through the use of a diaphorase/resazurin-coupled system. Inhibition of the IDH1 R132H enzymatic reaction leads to an increase in the amount of NADPH relative to a DMSO control and a concomitant increase in the fluorescence of the diaphorase/resazurin enzyme product resorufin at 590 nm. Compounds were screened as a concentration-titration series that ranged from 76 microM to 0.15 uM.

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: R03 DA032129
PI Name: Dr. Lenny Dang

Enzyme buffer was dispensed into black, solid 1536-well plates at 3 microL/well in 20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.001% Tween20, 0.05% BSA, 2 mM beta-ME and 5.2 nM IDH1 R132H. Then, 23 nL of compounds or DMSO were delivered to each well using a pin tool. The inherent compound fluorescence was measured at this point at 590 nm on a ViewLux plate reader (Ex 525, Em 598, bodipy filter). The substrate buffer (3 uL; 20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.001% Tween20, 0.05% BSA, 0.008 mM NADPH, and 1 mM a-ketoglutarate) was added to start the enzymatic reaction, and the plate was briefly spun at 270xg. After the reaction was allowed to progress for 80 minutes at room temperature, the remaining NADPH was detected with the diaphorase/resazurin-coupled system. Three microL of detection buffer (20 mM Tris buffer, pH 7.5, containing final concentrations of 10 mM MgCl2, 20 mM NaCl, 0.001% Tween20, 0.05% BSA, 0.53 uM diaphorase and 0.012 mM resazurin) was added, and after 5 minutes the fluorescence at 590 nm was measured on an Envision plate reader (Ex 544, Em 590, bodipy filter, 10 flashes). The %Activity was determined from the corrected fluorescence values. As no specific IDH1 R132H inhibitors have been identified in the literature, 1x (5.2 nM) and 0x IDH1 R132H enzyme controls (untreated) were included to normalize % Activity of identified inhibitors; 0x enzyme values corresponded to 100 % Activity (full inhibition), while 1x IDH1 R132H enzyme values were used to normalize 0 %A ctivity (no inhibition).

Concentration-response curves were fitted to the signals arising from the resulting fluorescence. The concentration-response curves were then classified based on curve quality (r2), response magnitude and degree of measured activity, and compounds were subsequently categorized based on their curve class. Active inhibitors showed concentration-dependent increases in fluorescence, concordant with a decrease in IDH1 R132H activity and less substrate NADPH utilization. Inactive compounds showed no effect on fluorescence signal relative to the DMSO control.

Keywords: Isocitrate dehydrogenase, IDH1, IDH1 R132H, 2-HG, 2-hydroxyglutarate, AML, glioma, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC

1. Ohgaki H: Epidemiology of brain tumors. Methods Mol Biol 2009, 472:323-342.
2. Dubbink HJ, Taal W, van Marion R, Kros JM, van Heuvel I, Bromberg JE, Zonnenberg BA, Zonnenberg CB, Postma TJ, Gijtenbeek JM et al: IDH1 mutations in low-grade astrocytomas predict survival but not response to temozolomide. Neurology 2009, 73(21):1792-1795.
3. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ et al: IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009, 360(8):765-773.
4. Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K, Koboldt DC, Fulton RS, Delehaunty KD, McGrath SD et al: Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009, 361(11):1058-1066.
5. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM, Fantin VR, Jang HG, Jin S, Keenan MC et al: Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature 2009, 462(7274):739-744.


Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: DA032129